The long-term objective of this research is to acquire a better understanding of de novo pyrimidine biosynthesis at the molecular level. This pathway is critical because the end products, the pyrimidine nucleotides, are necessary for DNA and RNA synthesis, molecules that contain the genetic information fundamental to growth, development and reproduction of all living organisms. Moreover, it has become a common target for anti-proliferation drugs. In particular, we are interested in the adaptation of this pathway and its enzymes to extreme conditions of temperature. Aspartate transcarbamoylase is the enzyme which catalyzes the second step of this pathway, the reaction between carbamoyl phosphate and aspartate to form carbamoyl aspartate, and is an important site of regulation in many organisms. In this proposal we focus on the enzyme aspartate transcarbamoylase from the thermophilic and barophilic archaeon Methanococcus jannaschii. We propose to carry out the structural analysis of the isolated catalytic subunit of this enzyme and its complex with the bisubstrate analogue N-phosphonacetyl-L-aspartate or other related substrate analogues using X-ray crystallography. This work will give insight into: (1) the molecular factors that impart thermostability to the catalytic subunit, (2) how the catalytic function adapts to high temperature, and (3) possibly substrate channeling. As enzymes of the pyrimidine pathway in hyperthermophilic archaea may serve as models for the assembly and function of the multienzymes in higher eukaryotes, the results from the present study may be relevant to human aspartate transcarbamoylase and provide the foundation for designing improved antineoplastic agents. The knowledge of the active site may assist in the design of inhibitors that bind to the active site. Better knowledge of substrate channeling in this system may be helpful for designing a new class of inhibitors disrupting the channeling of carbamoyl phosphate to the enzyme active site. Finally, understanding the means of extreme heat stability will allow us to engineer selected features in other aspartate transcarbamoylases to obtain enzymes with enhanced thermostability.